فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 32 (زمستان 1398)

Endophytic fungi are capable of synthesizing many plant secondary metabolites and as such make available new compounds for pharmaceutical investigation. Thus, the endophytic fungus Cr95 isolate was separated from the stem of Catharanthus roseus plant found in Iran and screened for the production of vinblastine. By considering its morphologic and genotypic properties, it became possible to identify this isolate, followed by grouping in the Chaetomium globosum species. Furthermore, the anti-proliferative activities of C. globosum Cr95 isolate against P. oryzae as a model fungus, was assessed. This endophytic fungus was screened for the production of vinca alkaloids using specific biochemical assays, tryptophan decarboxylase (TDC) encoding gene, and TLC and HPLC analyses. The endophytic isolate was found to have significant cytotoxic effects and showed the strongest anti-proliferative activity with an IC 50 value of 15.6 μg/ml. In the biochemical assays, C. globosum Cr 95 isolate showed a positive production of vinca alkaloids. TDC gene as the key enzyme in the terpenoid indole alkaloid biosynthetic pathway could be amplified from this isolate. The presence of vinblastine in fungal culture filtrate was confirmed through chromatographic and spectroscopic analyses, and the amount was estimated to be 78 μg/l. The cytotoxic activity of the partially purified fungal vinblastine against conidia of P. oryzae was evaluated using tetrazolium salt MTT assay, and maximum susceptibility was found to occur with the IC 50 value of 5 μg/ml. Fungal VBL in ethyl acetate extracts was characterized by TLC and HPLC, and its cytotoxic effects on P. oryzae were determined, which is indicative of its maximum susceptibility.To the best of the authors’ knowledge, this is the first report on vinblastine production from the endophytic fungus, C. globosum Cr 95 isolated from the C. roseus plant.

Although bacteria and archaea are able to grow and adapted to the petrol reservoirs during severalyears, there are no results from microbial diversity of oilfields with high temperature in Iran. Hence, the present study tried to identify microbial community in non-water flooding Zeilaei (ZZ) oil reservoir.

In this study, for the first time, non-water flooded high temperature Zeilaei oilfield was analyzed for its microbial community based on next generation sequencing of 16S rRNA genes.

The results obtained from this study indicated that the most abundant bacterial community belonged to phylum of Firmicutes (Bacilli) and Thermotoga, whileother phyla (Proteobacteria, Actinobacteria and Synergistetes) were much less abundant. Bacillus subtilis, B. licheniformis, Petrotoga mobilis, P. miotherma, Fervidobacterium pennivorans, and Thermotoga subterranea were observed with high frequency. In addition, the most abundant archaea were Methanothermobacter thermautotrophicus. Discussion and

Although there are many reports on the microbial community of oil filed reservoirs, this is the first report of large quantities of Bacillus spp. from a high temperature oil reservoir.

Poosti cheese is an artisanal cheese produced mainly from ewe’s milk in southwest Iran. The objective of this study was to identify lactobacilli isolated from Poosti cheese and to investigate their potential probiotic and antioxidative properties. Materials and methods 101 Gram-positive bacilli with catalase-negative reaction were isolated from Poosti cheese. Twenty isolates were selected based on their highest acid and bile salt tolerability. Using genus-specific primers, 10 isolates out of these were identified as Lactobacillus, and they were subsequently identified at species level using 16S rRNA gene sequencing. In vitro tests were used to assess their probiotic properties or antioxidative activities.

Six species were Lactobacillus plantarum, and the rest was Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus buchneri. The acid and bile tolerances were quantitatively close to the reference probiotic strain. L. plantarum S32E showed the highest survival under simulated gastric or intestinal conditions. Cholesterol reduction ability of L. plantarum S12E and L. acidophilus S37A was significantly higher than that of the reference strain. L. acidophilus S37A and L. plantarum S12E were more susceptible against common antibiotics. It was found that the highest antimicrobial activity was detected in the case of Listeria monocytogenes, and the extent of the activity was species- and strain-dependent. The best result of DPPH radical scavenging activity and hydroxyl radical scavenging activity was achieved by intact cells of L. paracasei S5D and by intracellular cell-free extracts of L. plantarum S8D, respectively. The results indicated that the superoxide dismutase activity of Lactobacillus could not be regarded as an appropriate predictor of antioxidative traits. The highest glutathione content belonged to L. acidophilus S37A and L. parcasei S5D. Discussion and

Overall, some of Lactobacillus strains of Poosti cheese could be considered as potential probiotic candidates; it should, however, be confirmed by further in vivo evaluation.

DNA polymerases, in addition to being indispensable in replication and repair, are also very useful in a number of molecular biology techniques such as DNA amplification, site-directed mutagenesis, DNA sequencing, different kinds of PCR, loop-mediated isothermal amplification (LAMP), etc. After the invention of PCR, efforts have been made to focus on the identification and isolation of thermo-tolerant enzymes that amplify DNA efficiently at high temperatures.

In this study, Geobacillus stearothermophilus strain10 was selected for the cloning of Bst DNA Pol I - encoding gene. Following DNA extraction from the bacterium, PCR was carried out to amplify the pol A gene using designed primers and to clone via pET32a expression vector followed by transfer to the heterologous E.coli BL21 host. The cloned gene was expressed by induction with IPTG and the resultant protein purified by IMAC column.

The activity of the functional fragment was assessed by LAMP and showed a relatively high DNA amplification ability in comparison with commercial Bst DNA Polymerase which is usually used in this amplification protocol. Discussion and

This study found thatKlenow fragment of recombinant Bst DNA Pol I can amplify uidA gene in E. coli ATCC25923 during the LAMP reaction. Separation of two fragments of the enzyme can improve the activity of Klenow fragment of enzyme in LAMP.

Acetoin production, by beneficial rhizobacteria, plays an important role in plant growth and resistance to plant pathogens. The aim of this work was to optimize the nutritional conditions using statistically based experimental designs for the production of B. subtilis GB03 acetoin.

Eight components of the medium and cultivation conditions were examined for their significance on the production of acetoin using the Plackett–Burman experimental design. Steepest ascent experiments were employed to approach the optimal region of the three factors and a central composite design was applied to determine their optimal levels.

Results indicated that glucose, ammonium phosphate and agitation speed had significant effects on the acetoin production. The significant medium components in our optimization medium were 75.91 g/l of glucose, 5.79 g/l of ammonium phosphate, and 213rpm of agitation speed. The maximum acetoin concentration of 26.1 g/l at 40 h was obtained. Volatiles emitted from bacteria on optimized medium showed phytotoxicity on Arabidopsis seedlings. Result revealed that 1 ppm of acetoin was the best for the promotion of plant growth. Discussion and

Statistical experimental designs appeared to be an influential tool for optimization of fermentation conditions to enhance the acetoin production. However, volatile components secreted from the bacteria in the optimized medium showed some degree of phytotoxicity on Arabidopsis seedlings.

Bacillus subtilis spore surface display technique has long been used to display antigens and enzymes for medical and industrial proposes. In this technique, the enzyme is genetically immobilized on the spore surface and one of the capabilities of spore displayed enzyme is the reusability. In this study, spore displayed tyrosinase was used for the bioconversion of soybean extracted daidzein to more hydroxylated, anticancer compound, 3ʹ-ODI.

Bacillus subtilis DB104 (pSDJH-cotE-tyr) which was constructed in our previous study was used as an enzyme source. The reaction was done in 37˚C for 1 hour. To detect the product of the reaction, high-performance liquid chromatography was used.

The results revealed that 1mM of daidzein was converted to about 1mM 3ʹ-ODI during 60 min by 4*108 spores. The retained activity of the spore displayed tyrosinase was also detected about 58% after three times usage. Discussion and

Spore surface displayed enzymes have created a new way to improve enzyme stability and reusability. Our results showed that active tyrosinase on the surface of the spores has the potential to be used in industrial conditions to produce more hydroxylated isoflavones.

Using the standard methods for the productions of unicellular algae have high harvest costs and in many cases is not affordable. Therefore, chemical fertilizers could be considered as an appropriate alternative for the production of unicellular algae. The green microalgae Haematococcus pluvialis is capable of producing high levels of astaxanthin and lipid in resistant cells called spore.

H. pluvialis with the initial density of 4.25 × 104 cell mL-1 transferred to the 3-liter containers. Fatty acid profiles, growth factors, chemical compositions and pigments of algae were investigated.

The results showed that the specific growth rate, the cell division per day, carbon dioxide fixation efficiency, biomass, and total biomass efficiency were significantly higher and lower in treatment 5 (G: I= 50:50) and 9 (Gf= 100), respectively. The highest protein (40.68%), lipid (34.88%) and carbohydrate (42.04%) contents were observed in treatments of 9, 2 (G: R= 50:50) and 8 (I= 100), respectively. The highest chlorophyll a (71.29 mg/g FW) in T9 and chlorophyll b (85.60 mg/g FW) in T3 (G: R= 75:25) and total carotenoid (206.11 mg/g FW) in T6 (G: R= 75:25). Saturated fatty acid was significantly highest in T6, but its poly unsaturated fatty acid was significantly highest in T2. Discussion and

The results indicated that the f/2 medium promotes biomass and lipid in T9 and T5, T6 made increase total carotene and SFA and T2 and T4 raised Mono saturated fatty acid and PUFA in H. pluvialis when it is cultured in brackish water.

Soft rot caused by pectolytic bacteria makes annual production and storage losses throughout the world. This study aimed to detect and identify pathogenic Pseudomonas causing soft rot in some of the vegetables and ornamentals.

During the growing seasons of 2015–17, plant samples of eggplant, maize, radiator plant, sweet pepper and tomato with water soak and soft rot symptoms were collected. Biochemical and morphological features were characterized according to the standard bacteriological criteria. The tuf encoding gene from these representatives was amplified using Bac-tuf-F and Bac-tuf-R primers, subjected to sequencing and aligned in the NCBI.

Total of 120 isolates were recovered from the samples. Based on synthesizing pectic enzymes, five putative Pseudomonas strains were selected. Based on the DNA sequence-based phylogeny, in combination with biochemical and morphological characteristics, these soft rot Pseudomonas were identified as Pseudomonas aeruginosa, P. entomophila, P. mosselii and P. putida. Koch's postulates were verified by re-isolating the strains from inoculated plant segments. Discussion and

To the best of our knowledge, this is the first evidence of the pathogenicity of P. aeruginosa on tomato, P. entomophila on sweet pepper and P. putida on eggplant. Furthermore, this study firstly reports the association of P. aeruginosa and P. mosselii with soft rot in maize and radiator plant, respectively.

To increase lactobacillus cells stability, different methods based on entrapment used and their effect on the culturability of entrapped cells was studied. Materials and methods In the present study, sub-lethal acid stress, biofilm-like structure, and the combination of plant gums were used to assess the culturability of entrapped Lactobacillus plantarum during different production phases.

Based on the obtained results, incubation of alginate entrapped cells in MRS broth containing acetic acid increased the survival rate by 29% compared to beads treated in MRS broth. In addition, incubation of alginate entrapped cells in acidic medium resulted in 5% higher survival of biofilm-like cells after the freeze-drying process. To increase the stability of cells during storage, cells were entrapped in alginate plus different plant gums. Incorporation of tragacanth gum and salep gum to alginate showed higher culturability of cells (4.6and 3.1 folds, respectively) in comparison to alginate treated beads. Additionally, the lowest inactivation constant rate (k value), 0.09, and highest D  value, 25 days, obtained for tragacanth gum at 4°C during storageindicating that treatment with tragacanth gum could preserve the cells better in comparison to other treatments under storage condition. Discussion and

The incubation of Lactobacillus plantarum beads in acidic MRS medium can result in increased culturability especially after freeze-drying. This can be due to cross-protection effect. Additionally, due to tragacanth gum traits, this plant gum could protect the entrapped cells better compared to other plant gums in the storage condition. In conclusion, we can use tragacanth gum as a second material for increasing the stability of entrapped L. plantarum cells.

Carbapenem-resistant Enterobacteriaceae (CRE) bacteria are difficult to treat because of their high antibiotic resistance levels that can be mediated by carbapenemase enzymes such as Klebsiella Pneumoniae Carbapenemase (KPC). The purposes of this study were to determine the genetic and resistance patterns and to detect of KPC enzyme in carbapenem-resistant strains of Enterobacteriaceae isolates.

In this study, antibiotic resistant pattern and genotyping of carbapenem-resistant Enterobacteriaceae isolates and frequency of KPC enzyme were investigated. During 16 months of conducting the study (December 2016 until April 2018), strains of Escherichia coli, Enterobacter spp. and Citrobacter spp. were isolated and identified from different clinical specimens and antibiotic susceptibility test was determined. In addition, the prevalence of the KPC enzyme was determined by PCR and two phenotypic methods including Modified Hodge Test (MHT) and the combination test by boronic acid. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was used for the determination of the clonal relationship of carbapenems resistant strains.

The results showed that among 520, 146 and 58 isolated of Escherichia coli, Enterobacter spp. and Citrobacter spp. effective antibiotics were carbapenems, piperacillin/tazobactam, amikacin, cefepime, fosfomycin, nitrofurantoin, and gentamicin, respectively. In addition, 12 strains (2.3%) of E. coli, 4 strains (2.7%) of Enterobacter and 4 strains (6.9%) of Citrobacter were resistant to carbapenems. The KPC investigation results showed the detection of this enzyme in 18 and 6 isolates using MHT and boronic acid methods respectively, but no producer strain was observed using PCR. The result of ERIC-PCR showed in carbapenem-resistant strains of Citrobacter, Enterobacter and Escherichia coli, 3, 4 and 9 distinct main clusters (patterns) were observed respectively. Discussion and

This study demonstrates the high antibiotic resistant prevalence and low specificity of standard phenotypic methods that were used for KPC detection in Isfahan City.

Pollution of agricultural soils with heavy metals is a serious problem in the world. The aim of this study was to produce a multipurpose biofertilizer which can remove cadmium from polluted soils as well as enhancing plant growth.

To study the ability of Pseudomonas putida strain PT for establishing an effective relationship with plants, the effects of some compounds present in plant root exudates were examined on chemotaxis, growth, and biofilm formation of bacteria. Pot experiments were performed by using biofertilizers prepared from P. putida PT by two different methods including seed immersion and immobilization of the bacterial cells on rice bran as carrier. After measuring the fresh weight and dry weight of each plant, cadmium concentrations in plant aerial tissues and soil in the presence and absence of biofertilizers was measured by atomic absorption spectrometry.

Results showed positive chemotactic responses of P. putida PT to sucrose, mannitol, glucose, alanine, histidine, tryptophan, succinic acid, malic acid, and citric acid. Succinic acid, mannitol, and sucrose promoted both biofilm formation and growth of P. putida PT. Malic acid enhanced only the bacterial growth, whereas histidine, tryptophan and glucose promoted biofilm formation. Both biofertilizers enhanced the fresh and dry weight of maize plants about 2-fold. The maximum reduction of cadmium concentration in the soil was observed in the presence of immobilized cells (97.57%), followed by the inoculation of bacterial cells by seed immersion method (68.67%). Cadmium concentration in plants was decreased from 6310 ppb in control experiment to 885 ppb and 2917.5 ppb in the presence of biofertilizers produced by cell immobilization and seed immersion techniques, respectively. Discussion and

Using these fertilizers is an approach to promote maize growth in cadmium contaminated soils along with decreasing metal concentrations in soil and plant.

Streptococcus pyogenes causes a variety of infectious and non-infectious diseases. Typing of S. pyogenes isolates is one of the essential tools in the epidemiological studies of this bacterium. Random Amplified Polymorphic DNA (RAPD) is a rapid, easy and inexpensive PCR-based typing technique. Low reproducibility of RAPD-PCR is the main disadvantage of this method which will be resolved by optimization of RAPD-PCR protocol.

In this study, optimization of RAPD-PCR protocolincluding DNA extraction method, primer type, concentrations of PCR reagents, and PCR program was performed using the factorial design of experiments for S. pyogenes ATCC 19615 as a standard strain. Then, sixteen S. pyogenes isolates were genotyped by using optimized protocol. Typability, reproducibility, and discriminatory power of the optimized protocol were examined.

Among three DNA extraction methods and seven primers that were used, modified set buffer DNA extraction method and P14 primer were selected, respectively. Optimum concentration of PCR reagents were 3 mM MgCl2, 150 pmol primers, 0.2 mM dNTPs, 10 ng template DNA, and 2 U Taq DNA polymerase and the optimum PCR program consisted of an initial denaturation for 4 min at 94°C followed by 45 cycles of 1 min at 94°C, 2 min at 31°C, 2 min at 72°C, and a final extension at 72°C for 10 min.Results of optimized RAPD-PCR were reproducible for S. pyogenes ATCC 19615 and all S. pyogenes isolates. Calculated discriminatory power was satisfactory (DI=1). Sixteen S. pyogenes isolates belonged to sixteen strains which were classified into 3 main clusters on a similarity level of 14%. Discussion and

A suitable and reproducible RAPD-PCR protocol was obtained for genotyping of S. pyogenes isolates using RAPD-PCR optimization. The optimized protocol in the present study can be used in subsequent experiments on RAPD-PCR profiling for epidemiological study of S. pyogenes isolates.

In recent years, numerous studies have been done on using biological approaches instead of chemical fertilizers and increasing sustainable production in agriculture systems. Some microorganisms can promote growth and yield in the plant. In this study, the effect of several plant-growth-promoting bacterial strains were evaluated on germination and seedling growth of four different plant species including sesame (Sasamum indicum), canola (Brassica napus), wheat (Triticum turgidum L.) and barley (Hordeum vulgar L.).

The seeds of four plant species were inoculated by five bacterial strains including Bacillus subtilis, Bacillus pumilus, Azotobacter chroococcum, Rhizobium meliloti and Stenotrophomonas. Their effects were evaluated on seed germination and growth parameters of seedlings under in vitro condition.

The results of this experiment showed that among gram-negative bacteria, the Stenotrophomonas sp. enhanced seed germination, root number, root length (cm), shoot length (cm), root fresh weight (g) and shoot fresh weight (g) significantly. Among the gram-positive strains, Basillus subtilis mostly promoted germination and the seedling growth. Discussion and

The effects of B. Pumilus on germination and seedling growth of cereals was evaluated first. The results indicated the potential application of Stenotrophomonas sp. on enhancement of plant seed germination, but the positive effects of it on oil seed crops was less than cereals seed. B. pumilus showed the most negative effect on germination of barely and canola.

Introduction A wide variety of oleaginous yeast and their productions have been used as dietary supplements in food and feed productions. Fatty acid profiles and lipid of oleaginous yeast strains were highly influenced by physic-chemical conditions of medium Material and method Pichia kudriavzevii isolated from the gill of pike perch was capable enough to produce a high quantity of lipid and polyunsaturated fatty acids (PUFA).One factor at time was used to investigate the effect of various selected substrates on growth and lipid production by Pichia kudriavzevii in batch fermentation, subsequently the key substrates were optimized for lipid, biomass, and phenolic components production by using response surface methodology. Results The results of RSM indicated that maximum lipid (28 percentage of dry weight biomass) and phenolic component (2.25 mg gallic acid /g of dry biomass) were achieved in media that had 60 g/l glucose and 9 g/l protein resources. Dry weight biomass which had the highest content of lipid produced the most phenolic components with the highest antioxidant activity (50 % Inhibition).Pichia kudriavzevii’ lipid mainly consisted of palmitic acid (21.7%), oleicacid (33.2%) and linoleic acid (23.2 %), pointing that these fatty acids could be considered a reliable source of biodiesel production. Discussion and conclusion The maximum content of phenolic components and lipid production were obtained in the same medium. To minimize the lipid oxidation, yeast was stimulated for high phenolic components production which had the highest antioxidant activity to prevent the accumulated lipid from oxidation. Overall, temperature reduction by varying methods had a significant impact on fatty acids profile of Pichia kudriavzevii lipid so that it could be regarded not only as biodiesel but also PUFAs sources.

In recent years, electrochemical detection techniques have proved quite promising since they are simple, fast and cost effective. Up to date, some electrochemical biosensors based on enzymes and microorganisms have been fabricated for the detection of phenol as a priority pollutant listed by the United States Environmental Protection Agency (USEPA). MWCNTs have been widely considered as attractive materials due to their high electrical conductivity, chemical stability and extremely high mechanical strength. The presented work includes the development of a fast, sensitive and miniaturized microbial conductometric biosensor for the determination of phenol based on the cells of Pseudomonas sp. (GSN23) and modified microelectrodes with MWCNTs.

Cells of Pseudomonas sp. (GSN23) were grown in the presence of phenol as the sole source of organic carbon and adapted cells were immobilized on the surface of gold interdigitated microelectrodes. Carbon nanotube (CNT) - modified microelectrodes were also prepared to test nanoparticle effect on the efficiency of biosensor performance.

From the results obtained with conductometric measurement, sensitive detection of phenol from 1 to 300 mg.L-1 (10-3187 µM), was estimated. Furthermore, substrate specificity and operational stability were investigated. Discussion and

The proposed system does not require any complex immobilization procedures and showed the linearity and repeatability with a high operational stability. The use of optimum amounts of MWCNTs and phenol adapted bacteria provide better sensor sensitivity by promoting the ions transfer within the structure of the biosensor

Cost-effective production of proteases is one of the challenges facing the enzyme industry. In this regard, there are no reports concerning the influence of low-intensity electric current and nanoparticles on microbial neutral proteases production. This study aimed at investigating the effect of these treatments on the neutral protease production of Aeromonas hydrophila MSB16.

The protease production of A. hydrophila MSB16 in the presence of ionic and non-ionic surfactants (SDS, Tween 80, Triton X-100, and Span 80), glycerol as well as various nanoparticles (Ag, Fe III, Fe (0), Al, Zn, and Cu), was investigated. Furthermore, the effect of an electric current (50 µA) exposure to the bacterial cells during the logarithmic and stationary phase for 10 min and 20 min was studied.

According to the results, electric current exposure to the bacterial cells entered the stationary phase for 10 min and 20 min increased the protease production of A. hydrophila MSB16 by 48.2% and 59.1%, respectively. However, this positive effect was not observed for log phase-bacterial cells. Besides, glycerol, Tween 80, Span 80 and Ag nanoparticles enhanced the protease production of A. hydrophila MSB16 by 56.4%, 40.4%, 24.8%, and 12.5%, respectively. Discussion and

Non-ionic surfactants, glycerol, and electric current increased the protease production of A. hydrophila MSB16. Interestingly, the influence of electric current on the protease production of A. hydrophila MSB16 was dependent on the growth phase of the bacterium.